* Dyson Perrins Laboratory, Oxford Centre for Molecular Sciences, South Parks Road, Oxford OX1 3QY, UK; The National Hellenic Research Foundation, 48, Vas, Constantinou Avenue, Athens 111635, Greece; Laboratory of Molecular Biophysics,... more
* Dyson Perrins Laboratory, Oxford Centre for Molecular Sciences, South Parks Road, Oxford OX1 3QY, UK; The National Hellenic Research Foundation, 48, Vas, Constantinou Avenue, Athens 111635, Greece; Laboratory of Molecular Biophysics, The Rex Richards ...
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Insect behaviour relies on an olfactory sensory system that controls a range of activities, from food choice and mating to oviposition, where pheromones play a central role. In Culex mosquitoes, egg-laying is accompanied by the release of... more
Insect behaviour relies on an olfactory sensory system that controls a range of activities, from food choice and mating to oviposition, where pheromones play a central role. In Culex mosquitoes, egg-laying is accompanied by the release of mosquito oviposition pheromone (MOP), which has been shown to affect the oviposition behaviour of conspecifics. Here, we investigated for the first time the effect of MOP on the oviposition rate of Culex pipiens biotype molestus, examining separately males and females, before and after mating and oviposition. Our results demonstrate that MOP is more likely to act as an oviposition stimulant rather than an attractant, since more gravid females laid eggs in its presence, while the number of male or female mosquitoes (virgin or mated) captured on pheromone-treated pots was similar to those treated with control water.
Research Interests: Physiology, Zoology, Biology, Insect Physiology, Medicine, and 6 moreMating, Sex Pheromone, Larva, Insect, Culex pipiens, and Pheromone
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The pH-dependence of sulphate-activated phosphorylase b has been studied in the direction of glycogen synthesis. The bell-shaped curve of the pH-dependence of the catalytic constant for the AMP-activated enzyme showed pK values of 6.1 and... more
The pH-dependence of sulphate-activated phosphorylase b has been studied in the direction of glycogen synthesis. The bell-shaped curve of the pH-dependence of the catalytic constant for the AMP-activated enzyme showed pK values of 6.1 and 7.3, but the curve for the enzyme activated by 0.9 M ammonium sulphate showed a drop of activity on the acid side at much higher pH values. Its bell was centred at pH 7.8 but it was too narrow to be characterized by only two pK values. The narrowness of the curve could be explained by positive co-operativity, but not its unusually steep acid side. We suggest that the fall on the acid side is due to more than one hydronation (addition of H+). The points can be fitted by a curve with two de-activating hydronations and a de-activating dehydronation having identical titration pK values of 7.5, and hence molecular values of 7.0, 7.5 and 8.0. If both 0.9 M ammonium sulphate and 5 mM AMP are added, the bell is as broad as with AMP alone, but is somewhat r...
Research Interests: Chemistry, Catalysis, Kinetics, Medicine, Biological Sciences, and 15 moreAnimals, Biochemical, Enzyme, CHEMICAL SCIENCES, Ammonium, Isoenzymes, Titration, Protein Conformation, Rabbits, Amino Acid Sequence, Hydrogen-Ion Concentration, Sulfates, Adenosine monophosphate, binding sites, and Medical and Health Sciences
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ABSTRACT Indirubin and indigo analogues were discovered as novel inhibitors of glycogen phosphorylase, a target to control hyperglycaemia in type 2 diabetes. We give a structural insight into the molecular basis of enzyme inhibition by... more
ABSTRACT Indirubin and indigo analogues were discovered as novel inhibitors of glycogen phosphorylase, a target to control hyperglycaemia in type 2 diabetes. We give a structural insight into the molecular basis of enzyme inhibition by indirubin-5-sulphonate, indirubin-3'-aminooxy-acetate, and indigo-5,5',7,7'-tetrasulphonate. The inhibitors bind at the inhibitor site, by intercalating between the side chains of Phe285 and Tyr613. In addition, two indirubin-3'-aminooxy-acetate molecules bind at the allosteric binding site and a new sub-site in the vicinity of the allosteric site. Comparative structural analyses indicate residues in the inhibitor and the allosteric sites that can be exploited to obtain more potent inhibitors.
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With an aim toward glycogenolysis control in Type 2 diabetes, we have investigated via kinetic experiments and computation the potential of indirubin (IC₅₀... more
With an aim toward glycogenolysis control in Type 2 diabetes, we have investigated via kinetic experiments and computation the potential of indirubin (IC₅₀ > 50 μM), indirubin-3'-oxime (IC₅₀ = 144 nM), KT5720 (K(i) = 18.4 nM) and staurosporine (K(i) = 0.37 nM) as phosphorylase kinase (PhKγtrnc) ATP-binding site inhibitors, with the latter two revealed as potent inhibitors in the low nM range. Because of lack of structural information, we have exploited information from homologous kinase complexes to direct in silico calculations (docking, molecular dynamics, and MMGBSA) to predict the binding characteristics of the four ligands. All inhibitors are predicted to bind in the same active site area as the ATP adenine ring, with binding dominated by hinge region hydrogen bonds to Asp104:O and Met106:O (all four ligands) and also Met106:NH (for the indirubins). The PhKγtrnc-staurosporine complex has the greatest number of receptor-ligand hydrogen bonds, while for the indirubin-3'-oxime and KT5720 complexes there is an important network of interchanging water molecules bridging inhibitor-enzyme contacts. The MM-GBSA results revealed the source of staurosporine's low nM potency to be favorable electrostatic interactions, while KT5720 has strong van der Waals contributions. KT5720 interacts with the greatest number of protein residues either by direct or 1-water bridged hydrogen bond interactions, and the potential for more selective PhK inhibition based on a KT5720 analogue has been established. Including receptor flexibility in Schrödinger induced-fit docking calculations in most cases correctly predicted the binding modes as compared with the molecular dynamics structures; the algorithm was less effective when there were key structural waters bridging receptor-ligand contacts.
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Research Interests: Chemistry, Kinetics, Protein Science, Protein, Medicine, and 12 moreMacromolecular X-Ray Crystallography, Glucose, Research article, Trehalose, Glycerol, Glycogen, Polyethylene Glycols, Ethylene Glycol, Biochemistry and cell biology, Phosphorylases, Adenosine monophosphate, and Molecular Sequence Data
ABSTRACT Malaria is the most deadly tropical disease, responsible for 863.000 deaths in 2008 (WHO, 2010). It is transmitted to humans by hematophagous mosquitoes, particularly Anopheles gambiae, carrying the pathogen parasite Plasmodium... more
ABSTRACT Malaria is the most deadly tropical disease, responsible for 863.000 deaths in 2008 (WHO, 2010). It is transmitted to humans by hematophagous mosquitoes, particularly Anopheles gambiae, carrying the pathogen parasite Plasmodium falciparum. Mosquito repellents are the most commonly used agents for prevention of infection by keeping infected mosquitoes away from their human targets. DEET (N, N-diethyl-m-toluamide) is the most widely used repellent worldwide, with a broad effectiveness against most insects (Moore SJ, 2007). However, health problems during long-term use and ineffectiveness a few hours after the application, indicates the necessity of new more effective repellents. Previous efforts to identify more efficient insect repellents were hindered by the absence of a known molecular target and were thus limited only to ligand-based QSAR approaches. Recently we have reported the high-resolution structure of AgamOBP1-DEET complex which provides the first example of a repellent recognized by an odorant binding protein (OBP) (Tsitsanou et al, 2011). One DEET molecule (Kd=31.3±1.4 μM), is bound with high shape complementarity to each subunit at the dimer interface and exploits 60 van der Waals interactions and one hydrogen bond to a water molecule. Moreover, we usedmolecularmodeling to predict the binding characteristics of 29 repellents. Our predictions were further confirmed by fluorescent displacement assays indicating that structure-based design can provide promising leads for the development of novel repellents. Given that it takes about 10 years to develop a new repellent, the rational structure-based design approach will play an increasing role in the search for more effective repellents.
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The crystal structures of free T-state and R-state glycogen phosphorylase (GP) and of R-state GP in complex with the allosteric activators IMP and AMP are reported at improved resolution. GP is a validated pharmaceutical target for the... more
The crystal structures of free T-state and R-state glycogen phosphorylase (GP) and of R-state GP in complex with the allosteric activators IMP and AMP are reported at improved resolution. GP is a validated pharmaceutical target for the development of antihyperglycaemic agents, and the reported structures may have a significant impact on structure-based drug-design efforts. Comparisons with previously reported structures at lower resolution reveal the detailed conformation of important structural features in the allosteric transition of GP from the T-state to the R-state. The conformation of the N-terminal segment (residues 7-17), the position of which was not located in previous T-state structures, was revealed to form an α-helix (now termed α0). The conformation of this segment (which contains Ser14, phosphorylation of which leads to the activation of GP) is significantly different between the T-state and the R-state, pointing in opposite directions. In the T-state it is packed bet...
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C-Glucopyranosyl imidazoles, thiazoles, and an N-glucopyranosyl tetrazole were assessed in vitro and ex vivo for their inhibitory efficiency against isoforms of glycogen phosphorylase (GP; a validated pharmacological target for the... more
C-Glucopyranosyl imidazoles, thiazoles, and an N-glucopyranosyl tetrazole were assessed in vitro and ex vivo for their inhibitory efficiency against isoforms of glycogen phosphorylase (GP; a validated pharmacological target for the development of anti-hyperglycaemic agents). Imidazoles proved to be more potent inhibitors than the corresponding thiazoles or the tetrazole. The most potent derivative has a 2-naphthyl substituent, a Ki value of 3.2 µM for hepatic glycogen phosphorylase, displaying also 60% inhibition of GP activity in HepG2 cells, compared to control vehicle treated cells, at 100 μM. X-Ray crystallography studies of the protein - inhibitor complexes revealed the importance of the architecture of inhibitor associated hydrogen bonds or sulfur σ-hole bond interactions to Asn284 OD1, offering new insights to structure-based design efforts. Moreover, while the 2-glucopyranosyl-tetrazole seems to bind differently from the corresponding 1,2,3-triazole compound, the two inhibitors are equipotent.
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ABSTRACT
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Research Interests: Chemistry, Kinetics, Enzyme Inhibitors, Medicine, Biological Sciences, and 15 moreComputer Simulation, Glucose, Mathematical Sciences, Ligand Binding, Drug Design, Phosphorylation, Free Energy, Active site, Perturbation Analysis, Hydrogen Atom, Ligands, Molecular Dynamic Simulation, Binding free energy, Binding affinity, and Decomposition Analysis
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A short synthesis of the spirohydantoin of glucopyranose 1β [a potent and specific inhibitor of glycogen phosphorylase], together with its inactive anomer 1α, from a readily available heptonolactone is described; this is the first... more
A short synthesis of the spirohydantoin of glucopyranose 1β [a potent and specific inhibitor of glycogen phosphorylase], together with its inactive anomer 1α, from a readily available heptonolactone is described; this is the first synthesis of a spirohydantoin of a pyranose in ...
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Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP... more
Human coilin interacting nuclear ATPase protein (hCINAP) directly interacts with coilin, a marker protein of Cajal Bodies (CBs), nuclear organelles involved in the maturation of small nuclear ribonucleoproteins UsnRNPs and snoRNPs. hCINAP has previously been designated as an adenylate kinase (AK6), but is very atypical as it exhibits unusually broad substrate specificity, structural features characteristic of ATPase/GTPase proteins (Walker motifs A and B) and also intrinsic ATPase activity. Despite its intriguing structure, unique properties and cellular localization, the enzymatic mechanism and biological function of hCINAP have remained poorly characterized. Here, we offer the first high-resolution structure of hCINAP in complex with the substrate ADP (and dADP), the structure of hCINAP with a sulfate ion bound at the AMP binding site, and the structure of the ternary complex hCINAP-Mg(2+) ADP-Pi. Induced fit docking calculations are used to predict the structure of the hCINAP-Mg(2+) ATP-AMP ternary complex. Structural analysis suggested a functional role for His79 in the Walker B motif. Kinetic analysis of mutant hCINAP-H79G indicates that His79 affects both AK and ATPase catalytic efficiency and induces homodimer formation. Finally, we show that in vivo expression of hCINAP-H79G in human cells is toxic and drastically deregulates the number and appearance of CBs in the cell nucleus. Our findings suggest that hCINAP may not simply regulate nucleotide homeostasis, but may have broader functionality, including control of CB assembly and disassembly in the nucleus of human cells.